Drought is a global threat that affects agricultural production. Plants have evolved several adaptive strategies to cope with drought. Stomata are essential structures for plants to control water status and photosynthesis rate. Stomatal closure is an efficient way for plants to reduce water loss and improve survivability under drought conditions. The opening and closure of stomata depend on the turgor pressure in guard cells. Three key signaling molecules, including abscisic acid (ABA), reactive oxygen species (ROS), and calcium ion (Ca²⁺), play pivotal roles in controlling stomatal closure. Plants sense the water-deficit signal mainly via leaves and roots. On the one hand, ABA is actively synthesized in root and leaf vascular tissues and transported to guard cells. On the other hand, the roots sense the water-deficit signal and synthesize CLAVATA3/EMBRYO-SURROUNDING REGION RELATED 25 (CLE25) peptide, which is transported to the guard cells to promote ABA synthesis. ABA is perceived by pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) receptors, which inactivate PP2C, resulting in activating the protein kinases SnRK2s. Many proteins regulating stomatal closure are activated by SnRK2s via protein phosphorylation. ABA-activated SnRK2s promote apoplastic ROS production outside of guard cells and transportation into the guard cells. The apoplastic H₂O₂ can be directly sensed by a receptor kinase, HYDROGEN PEROXIDE-INDUCED CA²⁺ INCREASES1 (HPCA1), which induces activation of Ca²⁺ channels in the cytomembrane of guard cells, and triggers an increase in Ca²⁺ in the cytoplasm of guard cells, resulting in stomatal closure. In this review, we focused on discussing the signaling transduction of ABA, ROS, and Ca²⁺ in controlling stomatal closure in response to drought. Many critical genes are identified to have a function in stomatal closure under drought conditions. The identified genes in the process can serve as candidate genes for genetic engineering to improve drought resistance in crops. The review summarizes the recent advances and provides new insights into the signaling regulation of stomatal closure in response to water-deficit stress and new clues on the improvement of drought resistance in crops.

Although seed vigor is a complex physiological trait controlled by quantitative trait loci, technological advances in the laboratory are being translated into applications for enhancing seed vigor in crop plants. In this article, we summarize and discuss pioneering work in the genetic modification of seed vigor, especially through the over-expression of protein L-isoaspartyl methyltransferase (PIMT, EC 2.1.1.77) in seeds. The impressive success in improving rice seed vigor through the over-expression of PIMT provides a valuable reference for engineering high-vigor seeds for crop production. In recent decades, numerous genes/proteins associated with seed vigor have been identified. It is hoped that such potential candidates may be used in the development of genetically edited crops for a high and stable yield potential in crop production. This possibility is very valuable in the context of a changing climate and increasing world population.

Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.

Drought and heat stress, especially their combination, greatly affect crop production. Many studies have described transcriptome, proteome and phosphoproteome changes in response of plants to drought or heat stress. However, the study about the phosphoproteomic changes in response of crops to the combination stress is scare. To understand the mechanism of maize responses to the drought and heat combination stress, phosphoproteomic analysis was performed on maize leaves by using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. Five-leaf-stage maize was subjected to drought, heat or their combination, and the leaves were collected. Globally, heat, drought and the combined stress significantly changed the phosphorylation levels of 172, 149 and 144 phosphopeptides, respectively. These phosphopeptides corresponded to 282 proteins. Among them, 23 only responded to the combined stress and could not be predicted from their responses to single stressors; 30 and 75 only responded to drought and heat, respectively. Notably, 19 proteins were phosphorylated on different sites in response to the single and combination stresses. Of the seven significantly enriched phosphorylation motifs identified, two were common for all stresses, two were common for heat and the combined stress, and one was specific to the combined stress. The signaling pathways in which the phosphoproteins were involved clearly differed among the three stresses. Functional characterization of the phosphoproteins and the pathways identified here could lead to new targets for the enhancement of crop stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of abiotic stressors.